The present invention relates to a cholesterol sensor enabling rapid and high accuracy quantification of cholesterol and cholesterol ester contained in a sample in a simplified manner.
A biosensor has been proposed in the Japanese Laid-Open Patent Publication Hei 2-062952 as a system for simplified quantification of a specific component in a sample without diluting or agitating a sample solution.
This prior art biosensor is completed by first forming an electrode system comprising a measuring electrode, a counter electrode and a reference electrode on an electrically insulating base plate by using a screen printing method or the like, and then forming an enzyme reaction layer comprising a hydrophilic polymer, oxidoreductase, an electron acceptor, and additionally a buffer, if occasion demands, in close proximity to the previously formed electrode system.
Upon dropping a sample solution containing a substrate on the enzyme reaction layer, dissolution of the layer takes place, which in turn triggers reaction between the enzyme and the substrate, causing a reduction of the electron acceptor. Upon completion of the enzyme reaction, the reduced electron acceptor is oxidized electrochemically. The concentration of the substrate in the sample can be determined by reading the oxidation current that flows upon electrochemical oxidation of the electron acceptor.
This biosensor can be used for measurements of various materials if an appropriate enzyme corresponding to the substrate of a target material is selected.
The use of cholesterol oxidase or cholesterol dehydrogenase as the oxidoreductase can yield a biosensor for measurement of serum cholesterol.
Serum cholesterol level which serves as diagnostic standard at various medical institutions is a sum of serum cholesterol and cholesterol ester concentrations.
As is known, cholesterol ester can not serve as a substrate for oxidation by cholesterol oxidase. Therefore, in order to determine serum cholesterol concentrations as diagnostic standard, an additional step of converting cholesterol ester into cholesterol becomes necessary. Cholesterol esterase is a known enzyme for catalyzing this step.
However, since cholesterol ester in sera is encapsulated in one kind of pseudomicelle lipoprotein, the probability of contact of this substance with the enzyme cholesterol esterase becomes low, which greatly prolongs conversion of cholesterol ester into cholesterol. It has been widely accepted that addition of an optimal surfactant can improve the probability of contact of cholesterol ester with cholesterol esterase, thereby increasing the catalytic activity of cholesterol esterase. For example, the Japanese Unexamined Patent Publication Hei 6-71440 proposes a structure of a cholesterol sensor for measuring cholesterol by coloring reaction where an enzyme having a cholesterol ester hydrolyzing activity coexists with a surfactant alkylphenoxypolyglycidol in the same layer.
However, inclusion of a surfactant in the biosensor having the structure as disclosed in the Japanese Laid-Open Patent Publication Sho 63-139242 or Hei 2-062952 results in marked inaccuracy of the sensor response probably due to the structure of the employed electrode system. In other words, the biosensor has a working electrode and a counter electrode whose surfaces are formed by printing a carbon paste. Whereas, the underlying layers and the leads of those electrodes are formed by printing a silver paste in order to improve their electric conductivity. When a sample solution dissolving a surfactant is brought into contact with the electrode system of this configuration, the surfactant invades the carbon layer to touch the silver leads of the underlying layer. In such a structure, the silver leads participate in the electrochemical reaction upon application of a potential onto the electrodes at the time of measurement of the responsive current value of the sensor, which sometimes causes an increase in the current value. This means that the resultant responsive current value of the sensor does not reflect exact cholesterol concentrations in the sample. Moreover, invasion of the surfactant into the carbon electrodes sometimes lowers electrode activity, i.e., electron conduction velocity in the electrode/liquid interface, which is considered to impair the response characteristic of the sensor.